Streptococcus pneumoniae exacts an enormous toll on humanity. This pernicious organism kills approximately 3700 people per day, the majority of whom are children below the age of five. The populations at greatest risk for suffering life-threatening infections are the elderly and the young, immunosuppressed and diabetic individuals, and those with hematologic malignancies and renal failure. In the United States, on a per annum basis, the organism is estimated to cause 3000 cases of meningitis, 50,000 cases of bacterimia, 500,000 cases of pneumoniae, and 7 million episodes of acute otitis media (inner ear infection) that result in one million doctor visits and 5 billion dollars in related expenses.
Mankind has long suffered the debilitating consequences of S. pneumoniae infection. The organism was first isolated by Louis Pasteur in 1881 and was identified as a primary cause of lobular pneumonia soon thereafter. There are now more than 90 distinct serotypes (46 serogroups), each with a different propensity to cause human disease. Our previous battles with this organism have taught us that our current strategies (vaccines and antibiotics) provide temporary solutions, rather than endpoints, for the problem. During the last century, multiple antibiotic classes were brought forward against the organism which responds, seemingly inevitably, using a combination of spontaneous mutagenesis, DNA transfer among related organisms, and amplification by positive selection, to circumvent the metabolic blockages set in place by the antibiotics. Once an antibiotic-resistant strain emerges, it spreads quickly via colonization-pneumococci are found in the nasopharynx of 15% of well adults and up to 65% of children in child-care settings. It is generally agreed that successful containment of this organism requires constant vigilance (large-scale antibiotic-resistance surveillance efforts are underway at the Centers for Disease Control), the stringent use of antibiotics, and a pipeline of drugs and vaccines that enable us to respond quickly to changes in the streptococcal population. Unfortunately, the development of new antibiotics is no longer considered economically feasible by the pharmaceutical industry. Hence, our pipelines are drying up even as strains resistant to our last-line antibiotics are beginning to appear. The current pharmaco-economic landscape suggests that the financial burden for future antibiotic development will fall primarily on the shoulders of governmental funding agencies in economically advantaged countries. The threat of domestic and foreign strains of multiple-drug-resistant S. pneumoniae continues to mount, and is extremely serious.
There is however, a molecular Achille's heal in S. pneumoniae: mevalonate kinase, the first enzyme in the mevalonate pathway harbors an allosteric site that can be used to switch-off isoprenoid biosynthesis, which is essential for growth of the organism. (Andreassi J L, 2nd, Dabovic K, Leyh T S. 2004. Streptococcus pneumoniae isoprenoid biosynthesis is downregulated by diphosphomevalonate: an antimicrobial target. Biochemistry 43: 16461-6) Diphosphomevalonate (DPM), an intermediate in the pathway, binds to the allosteric site with high affinity (Ki=400 nM) and reversibly inhibits the enzyme. S. pneumoniae mutants in which the mevalonate pathway has been inactivated are unable to survive in the mouse lung, and the levels of mevalonate in serum appear insufficient to support their growth. These important biological studies validate the mevalonate pathway as a target for antimicrobial research. Such studies also demonstrated that the human mevalonate kinase isozyme is not affected by the allostere (DPM)—it does not have a functioning allosteric site. The implication of these findings is that it may be possible to inhibit S. pneumoniae, and other low-G+C gram-positive streptococcal pathogens, with little influence on human metabolism.
The Mevalonate Pathway.
The pathway is comprised of three consecutive reactions (see FIG. 1) that are catalyzed by the enzymes mevalonate kinase (MK), phosphomevalonate kinase (PMK) and diphospho-mevalonate kinase decarboxylase (DPM-DC). The end-product of the pathway, isopentenyl diphosphate, is the 5-carbon building block used for the biosynthesis of isoprenoids, a diverse family of roughly 23,000 biologically active small molecules that includes cholesterol, steroid hormones, bile acids, electron transport carriers, carotenoids, vitamin A, Taxol, farnesyl diphosphate, and numerous other interesting compound classes. Given the ubiquity and metabolic significance of isoprenoids, it is not surprising that the mevalonate pathway is considered essential for the survival of organisms that require it for isoprenoid biosynthesis.
The GHMP Kinase Family.
MK, PMK and DPM-DC are each members of the GHMP-kinase superfamily. Continued study will provide mechanistic and structural comparisons across the superfamily that will enhance understanding of the catalytic machinery shared by all members of the family and the changes that enable both γ-phosphoryl transfer to different acceptors and addition of the decarboxylation chemistry. The superfamily began taking shape in 1988 when the three conserved sequence-motifs (I-III) used to recognize family members was first reported. Soon thereafter, the motifs were associated with the structural elements that carry out functions common to the family—ATP binding and γ-phosphoryl transfer. In 1993 the four “sugar kinases” from which the GHMP acronym derives (Galactokinase, Homoserine kinase, Mevalonate kinase and Phosphomevalonate kinase) were gathered together, forming the family. Currently, the Protein Families Database (Pfam) associates 841 unique protein sequences with the superfamily, approximately one-tenth of which do not yet have a defined function. The superfamily has evolved to produce nominally seven different catalysts (mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate decarboxylase, archeal shikimate kinase, galactokinase, homoserine kinase, and 4-(cytidine 5′-diphospho)-2c-methyl-d-erythritol kinase) each designed to transfer the γ-phosphoryl group of ATP to a different acceptor, and a putative transcription factor, Xol-1, that regulates development in C. elegans. Of the seven enzymes, six kinases and one decarboxylase, two have been linked to inherited human disease, two are involved in the biosynthesis of aromatic and non-aromatic amino acids, folates and ubiquinones, one is needed to deliver galactose to the glycolytic pathway, and four participate in either the mevalonate or non-mevalonate dependent synthesis of isoprenoids.
The relevance to human disease extends beyond the borders of antibiotic development. Mevalonic aciduria (MVA) and hyperimmunoglobulinemia D (HIDS), both orphan diseases, are caused by allelic changes in motif III of MK. HIDS, characterized by febrile attacks, abdominal pain, arthralgia and rashes, does not significantly reduce life expectancy, while the symptoms of MVA range from mild neurological disorder to progressive fatal disease and neonatal death. The MK A334T mutation found in MVA patients causes a 50-fold decrease in Vmax, resulting in a life-threatening increase in levels of mevalonate in plasma. The analogous mutation in the S. pneumoniae PMK causes similar effects, kcat decreases 63-fold. These two enzymes catalyze different reactions (transfer to a primary hydroxyl or phosphate monoester); yet, the catalytic consequences of homologous mutations in conserved regions of the superfamily are similar. Thus, the lessons gleaned from one family member do, in certain cases, extrapolate well to others. Galactokinase (GK) deficiency results in excess galactose and galactitol which causes cell-death in the lens epithelium, producing cataracts. Position 334 mutations in GK cause cataracts in humans, and the catalytic consequences of these mutations have not be determined. Position 334 is located in Motif I, it is chemically is well conserved, and structures of GK (P. furiosus) and PMK support that it performs similar catalytic functions across the family. Mutations in PMK at this position cause pronounced effects on the steady-state affinity of the non-nucleotide substrate, variable effects (tightening to weakening) on the nucleotide affinity, and small effects on kcat. These findings led to the prediction that the metabolic lesion resulting from the cataract-causing GK mutations is a decrease in the steady-state affinity of the enzyme for galactose. These examples demonstrate how catalytic paradigms, established using a representative member of a protein family, can provide testable hypotheses regarding the molecular etiology of disease.
The Protein Database (PDB) contains the structures of seven different members of the family. Sequence identity among the seven is low (10-20%) yet they share a great deal of three-dimensional similarity, the Cα RMSDs range from 2.6-4.0 Å. The structural scaffold has been well maintained over evolutionary time (particularly the ATP-binding and γ-phosphoryl transfer elements) while residues in the γ-phosphoryl acceptor pocket have been allowed to drift, sculpting active sites with altered specificity and, in the case of DPM-DC, additional chemistry.